PROJECT SUMMARY Fanconi Anemia (FA) is a rare autosomal recessive genetic disease characterized by congenital defects, bone marrow failure, cancer predisposition, and cellular hypersensitivity to DNA crosslinking agents (ICLs). FA is caused by mutations in one of 23 genes whose protein products collaborate in a pathway required for removing cytotoxic interstrand crosslinks (ICLs) from DNA. The D'Andrea laboratory has identified many of the molecular players and processes in the FA/BRCA pathway. In collaboration with the Soulier laboratory, we recently identified REV7 as the FA gene, FANCV. This was a key finding, since REV7/FANCV is emerging as a critical protein affecting several different DNA repair processes, by engaging with different binding partners through its C-terminal ?seatbelt? domain. When the seatbelt of REV7 is bound to SHLD3, it functions upstream in NHEJ. When the seatbelt of REV7 is bound to REV3, it functions as a translesion (TLS) polymerase, Pol?, required for bypassing an unhooked DNA crosslink, generated by the upstream FA proteins. Thus, REV7 deficiency, like the deficiency of other FA proteins, causes sensitivity to Mitomycin C (MMC), an ICL-inducing agent. Through preliminary studies for this grant, we identified novel REV7 interactors by IP-MS. Interestingly, we identified the binding partner, TRIP13, a novel ATPase which flips REV7 into an open conformation and releases REV3 and inactivates the FA/BRCA pathway. We also identified the REV7-binding protein SHLD2/FAM35A. Knockdown of this protein, like REV7 knockdown, disrupts the FA/BRCA pathway. The Specific Aims for the next five years of this grant are 1) to determine the role of the ATPase TRIP13 in the suppression of the FA/BRCA Pathway 2) to determine the role of the REV7/FANCV protein and its binding partner SHLD2/FAM35A in the activation of the FA/BRCA pathway 3) to employ knockout mouse models for REV7/FANCV and TRIP13 to evaluate the regulation of the FA/BRCA pathway.